Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or beta (β) cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent stem cells, such as, embryonic stem cells.
In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm.
By the end of gastrulation, the endoderm is partitioned into anterior-posterior domains that can be recognized by the expression of a panel of factors that uniquely mark anterior, mid, and posterior regions of the endoderm. For example, HHEX, and SOX2 identify the anterior region while CDX1, 2, and 4 identify the posterior region of the endoderm.
Migration of endoderm tissue brings the endoderm into close proximity with different mesodermal tissues that help in regionalization of the gut tube. This is accomplished by a plethora of secreted factors, such as FGFs, WNTS, TGF-βs, retinoic acid (RA), and BMP ligands and their antagonists. For example, FGF4 and BMP promote CDX2 expression in the presumptive hindgut endoderm and repress expression of the anterior genes Hhex and SOX2 (2000 Development, 127:1563-1567). WNT signaling has also been shown to work in parallel to FGF signaling to promote hindgut development and inhibit foregut fate (2007 Development, 134:2207-2217). Lastly, secreted retinoic acid by mesenchyme regulates the foregut-hindgut boundary (2002 Curr Biol, 12:1215-1220).
The level of expression of specific transcription factors may be used to designate the identity of a tissue. During transformation of the definitive endoderm into a primitive gut tube, the gut tube becomes regionalized into broad domains that can be observed at the molecular level by restricted gene expression patterns. The regionalized pancreas domain in the gut tube shows a very high expression of PDX1 and very low expression of CDX2 and SOX2. PDX1, NKX6.1, PTF1A, and NKX2.2 are highly expressed in pancreatic tissue; and expression of CDX2 is high in intestinal tissue.
Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Dorsal and ventral pancreatic domains arise from the foregut epithelium. Foregut also gives rise to the esophagus, trachea, lungs, thyroid, stomach, liver, and bile duct system.
Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene PDX1. In the absence of PDX1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, both exocrine and endocrine tissues arising from the differentiation of pancreatic endoderm.
D'Amour et al. describe the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005, 23:1534-1541; U.S. Pat. No. 7,704,738). Transplanting these cells under the kidney capsule of mice reportedly resulted in differentiation into more mature cells with characteristics of endodermal tissue (U.S. Pat. No. 7,704,738). Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX1 positive cells after addition of FGF10 and retinoic acid (U.S. Patent App. Pub. No. 2005/0266554). Subsequent transplantation of these pancreatic precursor cells in the fat pad of immune deficient mice resulted in the formation of functional pancreatic endocrine cells following a 3-4 months maturation phase (U.S. Pat. No. 7,993,920 and U.S. Pat. No. 7,534,608).
Fisk et al. report a system for producing pancreatic islet cells from human embryonic stem cells (U.S. Pat. No. 7,033,831). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of sodium butyrate and activin A (U.S. Pat. No. 7,326,572). The cells were then cultured with BMP antagonists, such as Noggin, in combination with EGF or betacellulin to generate PDX1 positive cells. The terminal differentiation was induced by nicotinamide.
Small molecule inhibitors have also been used for induction of pancreatic endocrine precursor cells. For example, small molecule inhibitors of TGF-β receptor and BMP receptors (Development 2011, 138:861-871; Diabetes 2011, 60:239-247) have been used to significantly enhance the number of pancreatic endocrine cells. In addition, small molecule activators have also been used to generate definitive endoderm cells or pancreatic precursor cells (Curr Opin Cell Biol 2009, 21:727-732; Nature Chem Biol 2009, 5:258-265).
HB9 (also known as H1XB9 and MNX1) is a BHLH transcriptional activator protein expressed early in pancreas development starting at approximately embryonic day 8. HB9 is also expressed in notochord and spinal cord. Expression of HB9 is transient and peaks at about day 10.5 in pancreatic epithelium being expressed in PDX1 and NKX6.1 expressing cells. At about day 12.5, HB9 expression declines and at later stages it becomes restricted only to β cells. In mice homozygous for a null mutation of HB9, the dorsal lobe of the pancreas fails to develop (Nat Genet. 23:67-70, 1999; Nat Genet. 23:71-75, 1999). HB9−/− β-cells express low levels of the glucose transporter, GLUT2, and NKX6.1. Furthermore, HB9−/− pancreas shows a significant reduction in the number of insulin positive cells while not significantly affecting expression of other pancreatic hormones. Thus, temporal control of HB9 is essential to normal β cell development and function. While not much is known about factors regulating HB9 expression in β cells, a recent study in zebrafish suggests that retinoic acid can positively regulate expression of HB9 (Development, 138, 4597-4608, 2011).
The thyroid hormones, thyroxine (“T4”) and triiodothyronine (“T3”), are tyrosine-based hormones produced by the thyroid gland and are primarily responsible for regulation of metabolism. The major form of thyroid hormone in the blood is T4, which has a longer half-life than T3. The ratio of T4 to T3 released into the blood is roughly 20 to 1. T4 is converted to the more active T3 (three to four times more potent than T4) within cells by deiodinase.
T3 binds to thyroid hormone receptors, TRα1 and TRβ1 (TR). TR is a nuclear hormone receptor, which heterodimerizes with retinoid X receptor. The dimers bind to the thyroid response elements (TREs) in the absence of ligand and act as transcriptional repressors. Binding of T3 to TR reduces the repression of TRE dependent genes and induces the expression of various target genes. While numerous studies have suggested a role for T3 in increasing β cell proliferation, reducing apoptosis, and improving insulin secretion, its role in cell differentiation is undefined.
Transforming growth factor β (“TGF-β”) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, fibrosis and specification of developmental fate. TGF-β superfamily members signal through a receptor complex comprising a type II and type I receptor. TGF-B ligands (such as activins, and growth differentiation factors (“GDF”s)) bring together a type II receptor with a type I receptor. The type II receptor phosphorylates and activates the type I receptor in the complex. There are five mammalian type II receptors: TβR-II, ActR-II, ActR-IIB, BMPR-II, and AMHR-II and seven type I receptors (ALKs 1-7). Activin and related ligands signal via combinations of ActR-II or ActR-IIB and ALK4 or ALK5, and BMPs signal through combinations of ALK2, ALK3, and ALK6 with ActR-II, ActR-IIB, or BMPR-II. AMH signals through a complex of AMHR-II with ALK6, and nodal has been shown to signal through a complex of ActR-IIB and ALK7 (Cell. 2003,113(6):685-700). Following binding of the TGF-B ligand to the appropriate receptor, the ensuing signals are transduced to the nucleus primarily through activation of complexes of Smads. Upon activation, the type I receptors phosphorylate members of the receptor-regulated subfamily of Smads. This activates them and enables them to form complexes with a common mediator Smad, Smad4. Smads 1, 5, and 8 are substrates for ALKs 1, 2, 3, and 6, whereas Smads 2 and 3 are substrates for ALKs 4, 5, and 7 (FASEB J 13:2105-2124). The activated Smad complexes accumulate in the nucleus, where they are directly involved in the transcription of target genes, usually in association with other specific DNA-binding transcription factors. Compounds that selectively inhibit the receptors for TGF-β, have been developed for therapeutic applications and for modulating cell fate in the context of reprogramming and differentiation from various stem cell populations. In particular, ALK5 inhibitors have been previously used to direct differentiation of embryonic stem cells to an endocrine fate (Diabetes, 2011, 60(1):239-47).
In general, the process of differentiating progenitor cells to functional β cells goes through various stages; and great strides have been made in improving protocols to generate pancreatic cells from progenitor cells such as human pluripotent stem cells. Despite these advances in research, each step in the process of differentiating progenitor cells presents a unique challenge. As such, there is still a need for a protocol resulting in functional endocrine cells and, in particular, functional β cells.